Kowalski, C. H.; Lawhorn, S.; Smith, T. J.; Corrigan, R. M.; Barber, M. F.

The microbiota can promote host health by inhibiting pathogen colonization, yet how host-resident fungi, or the mycobiota, contribute to this process remains unclear. The human skin mycobiota is uniquely stable compared to other body sites and dominated by yeasts of the genus Malassezia. We observe that colonization of human skin by Malassezia sympodialis significantly reduces subsequent colonization by the prominent bacterial pathogen Staphylococcus aureus. M. sympodialis secreted products possess potent bactericidal activity against S. aureus and are sufficient to impair S. aureus skin colonization. This bactericidal activity requires an acidic environment and is exacerbated by free fatty acids, demonstrating a unique synergy with host-derived epidermal defenses. Leveraging experimental evolution to pinpoint mechanisms of S. aureus adaptation in response to the skin mycobiota, we identified multiple mutations in the stringent response regulator Rel that promote survival against M. sympodialis. Similar Rel alleles have been reported in S. aureus clinical isolates, and natural Rel variants are sufficient for tolerance to M. sympodialis antagonism. Partial stringent response activation underlies tolerance to clinical antibiotics, with both laboratory-evolved and natural Rel variants conferring multidrug tolerance. These findings demonstrate the ability of the mycobiota to mediate pathogen colonization resistance, identify new mechanisms of bacterial adaptation in response to fungal antagonism, and reveal the potential for microbiota-driven evolution to shape pathogen antibiotic susceptibility.